Sepsis affects more than 300,000 individuals in the US annually, costs more than $16 billion/yr to treat, is associated with an acute mortality of 30%, and is expected to increase in incidence, making this disease a major public health concern. Care of these patients has remained primarily supportive despite intensive basic and clinical research efforts. Novel insights into its pathogenesis that can be applied to patient care are sorely needed. Our laboratory is studying the endothelium as a cell type that is injured in sepsis and that, in turn, mediates further damage to the host. Specifically, we are studying the angiopoietins a class of secreted proteins that act on endothelial cells through the Tie-2 receptor as regulators of vascular permeability in this disease. We have found that circulating angiopoietin-2 (Ang-2) is elevated in human subjects with severe sepsis, and that excess Ang-2 is sufficient to promote leak across endothelial cells and in otherwise healthy adult mice. More recently published studies of Ang-2 null mice have further validated Ang-2 as a mediator of increased permeability during inflammation. We have also found that angiopoietin-1 (Ang-1), which is similar in structure to Ang-2 but has opposite signaling effects, prevents vascular leak in murine endotoxemia. To achieve this remarkable protective effect, Ang-1 must activate a regulatory protein in the Rho-GTPase family called p190RhoGAP. In vivo depletion of p190RhoGAP is sufficient to abrogate the protection conferred by Ang-1 against endotoxin. These findings have led us to hypothesize that derangements in angiopoietins via excess Ang-2 and/or insufficient Ang-1 contribute to the marked hyperpermeability of sepsis. To explore this central question, we will apply the gold-standard model of rodent sepsis, cecal ligation and perforation, to Ang- 2 null mice, and wild-type mice treated either with a potent Ang-2 neutralizing antibody or with an Ang-1 expressing adenovirus to evaluate survival, permeability, and cellular responses (Aim 1). Given the unexpected ability of Ang-1 to counteract endotoxin-mediated permeability via p190RhoGAP activation, we will explore this mechanism of barrier defense further (Aim 2). Finally, we will return to human subjects with sepsis to perform a prospective study based in our emergency department to evaluate the predictive value of early changes in circulating Ang-2 and Ang-1 for death, shock, and other adverse outcomes (Aim 3). Aims 1 and 3 are highly translational, and Aim 2 will substantially elucidate endothelial barrier defense mechanisms in sepsis. Pilot data are strongly supportive of each aim, the team we have assembled has extensive experience in collaborative research (basic science, clinical research, pathology and epidemiology) in the area of sepsis, and testing each aim will be cost-efficient. These targeted studies will improve our understanding of the pathogenesis of sepsis, and potentially lead to novel diagnostic and therapeutic options for patients with severe sepsis. PUBLIC HEALTH RELEVANCE Sepsis is a common, costly, and lethal disease of increasing incidence; it may already account for 10% of all deaths in the US, making it a major public health threat whose impact is only likely to grow. Based on extensive preliminary data, we believe that angiopoietins may be critically involved in the pathogenesis of this disease. The proposed experiments will enable us to evaluate the diagnostic and therapeutic potential of these proteins in sepsis.